DESIGN AND OPTIMIZATION OF A BIOSENSOR FOR THE DETECTION AND QUANTIFICATION OF T-LYMPHOCYTES
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In health care, cell enumeration by immunophenotyping has been crucial in diagnosing and treating patients with HIV, leukemia, or bone marrow transplantation. One key use of immunophenotyping is for detection of HIV. The increase in the number of HIV in blood leads to a significant drop in one subset of leukocyte, the T helper cells (CD4 cells), in the patient?s blood. Detection and quantification of CD4 cells are thus important measures for the indication of HIV infection. Many immunophenotyping techniques have been developed in the past but the flow cytometry has stood out as the golden standard. Flow cytometric immunophenotyping uses the receptor proteins present on the T-helper cells, CD3, CD4, and CD45 for quantification. This technique, however, is very costly and not affordable to all health care sectors. This project investigates a design and optimization of a biosensor that quantifies the amount of T-lymphocytes in human blood. Quantification of T lymphocytes is useful as a positive control in the case of HIV detection and as a determinant of immunological status of the patient during immunosuppressive therapy. A microfluidic biosensor based on liposome signal generation and amplification is adapted to the detection of the T-lymphocytes using two specific sets of antibodies recognizing CD3 and CD45 proteins on the surface of the cells. This microfluidic biosensor utilizes superparamagnetic beads tagged with CD45 antibody as capture probe and sulforhodamine B entrapping immunoliposomes tagged with CD3 antibody as reporter probe. The assay condition is optimized for the running buffer composition and the flow time resulting in assay times of approximately 10 minutes. Non-specific interactions of the liposomes and beads are measured and reduced by approximately 30% using a blocking solution. The non-specific interactions of the red blood cells are reduced by lysing the red blood cell prior to testing. A dose response curve of varying human blood concentrations is created to show the correlation of the signal with respect to different concentrations of cells. The limit of detection is determined to be 80-220 T-lymphocytes. The micro-biosensor is designed to be a simple and easy to use device that can be manufactured and operated at low cost and was thus applicable for testing in resource-limited settings. It has a great potential as a tool for detection and quantification of T-lymphocytes since it uses the same biological recognition principles as used in flow cytometry, however, its simplicity and low cost make it an attractive alternative .
dissertation or thesis